The detection of differentially expressed genes in pre-invasive breast tissue, specifically in non-comedo ductal carcinoma in situ as compared to genes expressed in normal tissue, is useful in the diagnosis, prognosis and treatment of human breast cancer. Such differentially expressed genes are effective marker genes indicating the significantly increased risk of breast cancer in a patient expressing these differentially expressed marker genes. These marker genes are useful in the detection, early diagnosis, and treatment of breast cancer in humans.
The discovery of the function of the BRCA 1 gene has broad utility including, in the present invention, development of methods to treat familial and sporadic breast cancers as well as screen for therapeutic drugs through production of important indicator compounds.
Of the differentially expressed genes described in this invention, DCIS-1 encodes a gene similar to the M2 subunit of hamster ribonucleotide reductase. The M2 subunit of ribonucleotide reductase (RibRed, hereafter) is responsible for regulation of RibRed. The differential levels of expression of the marker genes described in this invention (Seq ID No.s 1-7), indicate genetic changes which have been linked to the presence of pre-invasive breast cancer.
The BRCA1 gene (Seq. ID No. 47) is differentially expressed in invasive breast cancer cells. The BRCA1 gene product is a negative regulator of mammary cell proliferation which is expressed at diminished levels in sporadic breast cancer.
1. Technical Field
The present invention relates generally to methods of detection and diagnosis of breast cancer and more particularly to a diagnostic method which relies on the identification of marker genes expressed in pre-invasive cancers by microscopically-directed cloning. Furthermore, this invention concerns the prevention, detection, and diagnosis of breast cancer by addressing the molecular events which occur during the earliest alterations in breast tissue.
The present invention also relates generally to methods of treatment of breast cancer, and more particularly to gene therapy methods and methods for screening compounds that induce expression of the BRCA1 gene product.
2. Description of the Prior Art
It will be appreciated by those skilled in the art that there exists a need for a more sensitive and less invasive method of early detection and diagnosis of breast cancer than those methods currently in use. Breast cancer presents inherent difficulties in regard to the ease with which it is detected and diagnosed. This is in contrast to detection of some other common cancers, including skin and cervical cancers, the latter of which is based on cytomorphologic screening techniques.
There have been several attempts to develop improved methods of breast cancer detection and diagnosis. In the attempts to improve methods of detection and diagnosis of breast cancer, numerous studies have searched for oncogene mutations, gene amplification, and loss of heterozygosity in invasive breast cancer (Callahan, et al., 1992; Cheickh, et al., 1992; Chen, et al, 1992; and, Lippman, et al, 1990). However, few studies of breast cancer have analyzed gene mutations and/or altered gene expression in ductal carcinoma in situ (DCIS). Investigators have demonstrated high levels of p53 protein in 13-40% of DCIS lesions employing a monoclonal antibody to p53, and subsequent sequencing demonstrated mutations in several cases (Poller et al, 1992). The neu/erbB2 gene appears to be amplified in a subset of DCIS lesions (Allred et al, 1992; Maguire et al, 1992). Histologic analysis of DCIS cases suggests that mutations and altered gene expression events, as well as changes in chromatin and DNA content, occur predominantly in comedo DCIS (Bxc3x6cker et al, 1992; Killeen et al, 1991; and, Komitowski et al, 1990), which has a rapid rate of local invasion and progression to metastasis. Thus, there are presently no reliable marker genes for non-comedo DCIS (NCDCIS, hereafter).
Cancer in humans appears to be a multi-step process which involves progression from pre-malignant to malignant to metastatic disease which ultimately kills the patient. Epidemiologic studies in humans have established that certain pathologic conditions are xe2x80x9cpre-malignantxe2x80x9d because they are associated with increased risk of malignancy. There is precedent for detecting and eliminating pre-invasive lesions as a cancer prevention strategy: dysplasia and carcinoma in-situ of the uterine cervix are examples of pre-malignancies which have been successfully employed in the prevention of cervical cancer by cytologic screening methods. Unfortunately, because the breast cannot be sampled as readily as cervix, the development of screening methods for breast pre-malignancy involves more complex approaches than cytomorphologic screening now currently employed to detect cervical cancer.
Pre-malignant breast disease is also characterized by an apparent morphological progression from atypical hyperplasias, to carcinoma in-situ (pre-invasive cancer) to invasive cancer which ultimately spreads and metastasizes resulting in the death of the patient. Careful histologic examination of breast biopsies has demonstrated intermediate stages which have acquired some of these characteristics but not others. Detailed epidemiological studies have established that different morphologic lesions progress at different rates, varying from atypical hyperplasia (with a low risk) to comedo ductal carcinoma-in-situ which progresses to invasive cancer in a high percentage of patients (London et al, 1991; Page et al, 1982; Page et al, 1985; Page et al, 1991; and Page et al, 1978). Family history is also an important risk factor in the development of breast cancer and increases the relative risk of these pre-malignant lesions (Dupont et al, 1985; Dupont et al, 1993; and, London et al, 1991). Of particular interest is non-comedo carcinoma-in-situ which is associated with a greater than ten-fold increased relative risk of breast cancer compared to control groups (Ottesen et al, 1992; Page et al, 1982). Two other reasons besides an increased relative risk support the concept that DCIS is pre-malignant: 1) When breast cancer occurs in these patients it regularly occurs in the same region of the same breast where the DCIS was found; and 2) DCIS is frequently present in tissue adjacent to invasive breast cancer (Ottesen et al, 1992; Schwartz et al, 1992). For these reasons DCIS very likely represents a rate-limiting step in the development of invasive breast cancer in women.
DCIS (sometimes called intraductal carcinoma) is a group of lesions in which the cells have grown to completely fill the duct with patterns similar to invasive cancer, but do not invade outside the duct or show metastases at presentation. DCIS occurs in two forms: comedo DCIS and non-comedo DCIS. Comedo DCIS is often a grossly palpable lesion which was probably considered xe2x80x9ccancerxe2x80x9d in the 19th and early 20th century and progresses to cancer (without definitive therapy) in at least 50% of patients within three years (Ottesen et al, 1992; Page et al, 1982). Most of the molecular alterations which have been reported in pre-malignant breast disease have been observed in cases of comedo DCIS (Poller et al, 1993; Radford et al, 1993; and, Tsuda et al, 1993). Non-comedo DCIS is detected by microscopic analysis of breast aspirates or biopsies and is associated with a 10 fold increased risk of breast cancer, which corresponds to a 25-30% absolute risk of breast cancer within 15 years (Ottesen et al, 1992; Page et al, 1982; and, Ward et al, 1992).
Widespread application of mammography has changed the relative incidence of comedo and non-comedo DCIS such that NCDCIS now represents the predominant form of DCIS diagnosed in the United States (Ottesen et al, 1992; Page et al, 1982; and Pierce et al, 1992). Both forms of DCIS generally recur as invasive cancer at the same site as the pre-malignant lesion (without definitive therapy). The precursor lesions to DCIS are probably atypical ductal hyperplasia and proliferative disease without atypia which are associated with lower rates of breast cancer development, but show further increased risk when associated with a family history of breast cancer (Dupont et al, 1985; Dupont et al, 1989; Dupont et al, 1993; Lawrence, 1990; London et al, 1991; Page et al, 1982; Page et al, 1985; Page et al, 1991; Page et al, 1978; Simpson et al, 1992; Solin et al, 1991; Swain, 1992; Weed et al, 1990).
What is needed, then, is a sensitive method of detection and diagnosis of breast cancer when the cancerous cells are still in the pre-invasive stage. To illustrate the usefulness in early breast cancer detection of a marker gene and its encoded protein, consider the dramatic impact that prostate specific antigen has had on early stage prostate cancer. This method of early detection and diagnosis of breast cancer is presently lacking in the prior art.
Breast cancer occurs in hereditary and sporadic forms. Recently the BRCA 1 gene has been cloned and shown to be mutated in kindreds with hereditary breast and ovarian cancer (Hall et al. 1990, Miki, Y. et al. 1994, Friedman et al. 1994, Castilla et al. 1994, Simard et al. 1994). Although 92% of families with two or more cases of early-onset breast cancer and two cases of ovarian cancer have germ-line mutations in BRCA 1 (Narod et al. in press), the gene has not been shown to be mutated in any truly sporadic case to date (Futreal et al. 1994). Despite the surprising paucity of somatically acquired mutations in sporadic breast cancer, it is still a likely tumor suppressor gene with a key role in breast epithelial cell biology. The BRCA 1 gene encodes a protein of 1863 amino acids with a predicted zinc finger domain observed in proteins which regulate gene transcription. Until the discovery of the function of the BRCA1 gene in conjuction with the development of the present invention, the function was unknown.
Epidemiologic studies have established that NCDCIS of the breast is associated with a ten-fold increased risk of breast cancer (absolute risk of 25-30%). It seems likely that this pre-invasive lesion is a determinate precursor of breast cancer because the subsequent development of breast cancer is regularly in the same region of the same breast in which the NCDCIS lesion was found. Important aspects of the present invention concern isolated DNA segments and those isolated DNA segments inserted into recombinant vectors encoding differentially expressed marker genes in abnormal tissue, specifically in NCDCIS, as compared with those expressed in normal tissue, and the creation and use of recombinant host cells through the application of DNA technology, which express these differentially expressed marker genes (Sambrook et al, 1989).
Because there are no cell lines or animal models which clearly display known characteristics of pre-invasive breast disease, human breast tissue samples are essential for studying pre-invasive breast disease. Using human tissue samples, we subsequently have developed a method for cDNA cloning from histologically identified lesions in human breast biopsies. We have used this method to clone genes which are differentially expressed in pre-invasive breast lesions such as NCDCIS lesions as compared to genes expressed in normal tissue. The differentially expressed genes detected in pre-invasive breast cancer are called marker genes. Identification of marker genes for pre-invasive breast disease provides improved methods for detection and diagnosis of pre-invasive breast cancer tissue, and further provides marker genes for studies of the molecular events involved in progression from pre-invasive to malignant breast disease.
Analysis of marker gene expression in NCDCIS presents the advantage that cancerous breast tissue at that stage is non-invasive. Detection and diagnosis of NCDCIS by means of differentially expressed marker genes compared to the same marker genes in normal breast tissue, would allow a greater ability to detect, prevent and treat the disease before it becomes invasive and metastasizes. The stage or intermediate condition of NCDCIS is a particularly good candidate for early intervention because it is 1) prior to any invasion and thus prior to any threat to life; 2) it is followed by invasive carcinoma in over 30% of cases if only treated by biopsy; and, 3) there is a long xe2x80x9cwindowxe2x80x9d of opportunity (4-8 years) approximately before invasive neoplasia occurs. Thus, NCDCIS is an ideal target for early diagnosis. While these morphologically defined intermediate endpoints have been widely accepted, progress in defining the molecular correlates of these lesions has been hampered by an inability to identify and sample them in a manner which would allow the application of molecular techniques.
Frozen tissue blocks from breast biopsies were used to construct and screen cDNA libraries prepared from NCDCIS tissue, normal breast tissue, breast cancer tissue, and normal human breast epithelial cells. Several cDNAs which were differentially expressed in human DCIS epithelial cells compared to normal breast epithelial cells were cloned and sequenced. One gene which is differentially expressed is the M2 subunit of RibRed which is expressed at low levels in human breast epithelial cells but at higher levels in 4 out of 5 DCIS tissue samples. It is presumed that the altered morphologic appearance and determinant biologic behavior of DCIS results from altered expression of genes (such as RibRed) which is important in the induction of breast cancer in humans.
This invention, therefore, provides a method of detecting and diagnosing pre-invasive breast cancer by analyzing marker genes which are differentially expressed in non-comedo DCIS cells. Histopathologic studies have demonstrated that these morphologic patterns in breast tissue lead to invasive breast cancer in at least 20-30% of patients. The present method analyzes gene expression in normal, pre-malignant and malignant breast biopsies; and, it allows simultaneous comparison and cloning of marker genes which are differentially expressed in pre-invasive breast cancer. These marker genes can then be used as probes to develop other diagnostic tests for the early detection of pre-invasive breast cancer.
The present invention concerns DNA segments, isolatable from both normal and abnormal human breast tissue, which are free from total genomic DNA. The isolated DCIS-1 protein product is the regulatory element of the RibRed enzyme. This and all other isolatable DNA segments which are differentially expressed in preinvasive breast cancer can be used in the detection, diagnosis and treatment of breast cancer in its earliest and most easily treatable stages. As used herein, the term xe2x80x9cabnormal tissuexe2x80x9d refers to pre-invasive and invasive breast cancer tissue, as exemplified by collected samples of non-comedo or comedo DCIS tissues.
As used herein, the term xe2x80x9cDNA segmentxe2x80x9d refers to a DNA molecule which has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment encoding a differentially expressed protein (as measured by the expression of mRNA) in abnormal tissue refers to a DNA segment which contains differentially expressed-coding sequences in abnormal tissue as compared to those expressed in normal tissue, yet is isolated away from, or purified free from, total genomic DNA of Homo sapiens sapiens. Furthermore, a DNA segment encoding a BRCA1 protein refers to a DNA segment which contains BRCA1 coding sequences, yet is isolated away from, or purified free from, total genomic DNA of Homo sapiens sapiens. Included within the term xe2x80x9cDNA segmentxe2x80x9d, are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like.
Similarly, a DNA segment comprising an isolated or purified differentially expressed gene or comprising an isolated or purified BRCA1 gene refers to a DNA segment including differentially expressed coding sequences or BRCA1 coding sequences isolated substantially away from other naturally occurring genes or protein encoding sequences. In this respect, the term xe2x80x9cgenexe2x80x9d is used for simplicity to refer to a functional protein, polypeptide or peptide encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences and cDNA sequences. xe2x80x9cIsolated substantially away from other coding sequencesxe2x80x9d means that the gene of interest, in this case, any differentially expressed marker gene or the BRCA1 gene, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.
In particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences which encode differentially expressed genes in pre-invasive breast cancer, each which includes within its amino acid sequence an amino acid sequence in accordance with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, all seq id no:s 1-7 are derived from non-comedo DCIS samples from Homo sapiens sapiens. In other particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences which encode the M2 subunit of human RibRed that includes within its amino acid sequence the similar amino acid sequence of hamster RibRed corresponding to the M2 subunit of hamster RibRed.
In certain embodiments, the invention concerns isolated DNA segments and recombinant vectors which partially or wholly encode a protein or peptide that includes within its amino acid sequence an amino acid sequence essentially as partially or wholly encoded, respectively, by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. Naturally, where the DNA segment or vector encodes a full length differentially expressed protein, or is intended for use in expressing the differentially expressed protein, the most preferred sequences are those which are essentially as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 and which encode a protein that exhibits differential expression, e.g., as may be determined by the differential display or differential sequencing assay, as disclosed herein.
The term xe2x80x9ca sequence essentially as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7xe2x80x9d means that the sequence substantially corresponds to a portion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, respectively, and has relatively few nucleotides which are not identical to, or a biologically functional equivalent of, the nucleotides of the respective SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. The term xe2x80x9cbiologically functional equivalentxe2x80x9d is well understood in the art and is further defined in detail herein, for example see pages 24 through 25. Accordingly, sequences which have between about 70% and about 80%; or more preferably, between about 81% and about 90%; or even more preferably, between about 91% and about 99%; of amino acids which are identical or functionally equivalent to the amino acids of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 will be sequences which are xe2x80x9cessentially as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7xe2x80x9d, respectively.
In particular embodiments, the invention concerns a drug screening method and a gene therapy method that use isolated DNA segments and recombinant vectors incorporating DNA sequences which encode a protein that includes within its amino acid sequence an amino acid sequence in accordance with SEQ ID NO:49, SEQ ID NO:49 derived from breast tissue from Homo sapiens. In other particular embodiments, the invention concerns isolated DNA sequences and recombinant DNA vectors incorporating DNA sequences which encode a protein that includes with its amino acid sequence the amino acid sequence of the BRCA1 gene product from human breast tissue.
In certain embodiments, the invention concerns methods using isolated DNA segments and recombinant vectors which partially or wholly encode a protein or peptide that includes within its amino acid sequence an amino acid sequence essentially as set forth in SEQ ID NO:49. Naturally, where the DNA segment or vector encodes a full length BRCA1 protein, or is intended for use in expressing the BRCA1 protein, the most preferred sequences are those which are essentially as set forth in SEQ ID NO:47 and which encode a protein that retains activity as a negative growth regulator in human breast cells, as may be determined by antisense assay, as disclosed herein.
The term xe2x80x9ca sequence essentially as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7xe2x80x9d means that the sequence substantially corresponds to a portion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, respectively, and has relatively few nucleotides which are not identical to, or a biologically functional equivalent of, the nucleotides of the respective SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. The term xe2x80x9cbiologically functional equivalentxe2x80x9d is well understood in the art and is further defined in detail herein, for example see pages 24 through 25. Accordingly, sequences which have between about 70% and about 80%; or more preferably, between about 81% and about 90%; or even more preferably, between about 91% and about 99%; of amino acids which are identical or functionally equivalent to the amino acids of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 will be sequences which are xe2x80x9cessentially as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7xe2x80x9d, respectively.
The term xe2x80x9ca sequence essentially as set forth in SEQ ID NO:49xe2x80x9d means that the sequence substantially corresponds to a portion of SEQ ID NO:49 and has relatively few amino acids which are not identical to, or a biologically functional equivalent of, the nucleotides of SEQ ID NO:49. The term xe2x80x9cbiologically functional equivalentxe2x80x9d is well understood in the art and is further defined in detail herein, for example see pages 24 through 25. Accordingly, sequences which have between about 70% and about 80%; or more preferably, between about 81% and about 90%; or even more preferably, between about 91% and about 99%; of amino acids which are identical or functionally equivalent to the amino acids of SEQ ID NO:49 will be sequences which are xe2x80x9cessentially as set forth in SEQ ID NO:49xe2x80x9d.
In certain other embodiments, the invention concerns isolated DNA segments and recombinant vectors that include within their sequence a nucleic acid sequence essentially as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7. The term xe2x80x9cessentially as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7xe2x80x9d is used in the same sense as described above and means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, and has relatively few codons which are not identical, or functionally equivalent, to the codons of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively. Again, DNA segments which encode proteins exhibiting differential expression will be most preferred. The term xe2x80x9cfunctionally equivalent codonxe2x80x9d is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids (see FIG. 8).
In certain other embodiments, the invention concerns a method for screening drugs and a gene therapy method which involve the use of isolated DNA segments and recombinant vectors that include within their sequence a nucleic acid sequence essentially as set forth in SEQ ID NO:47 and SEQ ID NO:48. The term xe2x80x9cessentially as set forth in SEQ ID NO:47 and SEQ ID NO:48xe2x80x9d is used in the same sense as described above and means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO:47 and SEQ ID NO:48 respectively, and has relatively few codons which are not identical, or functionally equivalent, to the codons of SEQ ID NO:47 and SEQ ID NO:48, respectively. Again, DNA segments which encode proteins exhibiting the negative regulatory activity of the BRCA1 will be most preferred. The term xe2x80x9cfunctionally equivalent codonxe2x80x9d is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids (see FIG. 8).
It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5xe2x80x2 or 3xe2x80x2 sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences which may, for example, include various non-coding sequences flanking either of the 5xe2x80x2 or 3xe2x80x2 portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.
Excepting intronic or flanking regions, and allowing for the degeneracy of the genetic code, sequences which have between about 20% and about 50%; or more preferably, between about 50% and about 70%; or even more preferably, between about 70% and about 99%; of nucleotides which are identical to the nucleotides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7 will be sequences which are xe2x80x9cessentially as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7xe2x80x9d, respectively. Sequences which are essentially the same as those set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7 may also be functionally defined as sequences which are capable of hybridizing to a nucleic acid segment containing the complement of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, under relatively stringent conditions. Suitable relatively stringent hybridization conditions will be well known to those of skill in the art (Sambrook et al, 1989).
Excepting intronic or flanking regions, and allowing for the degeneracy of the genetic code, sequences which have between about 20% and about 50%; or more preferably, between about 50% and about 70%; or even more preferably, between about 70% and about 99%; of nucleotides which are identical to the nucleotides of SEQ ID NO:47 and SEQ ID NO:48 will be sequences which are xe2x80x9cessentially as set forth in SEQ ID NO:47 and SEQ ID NO:48xe2x80x9d, respectively. Sequences which are essentially the same as those set forth in SEQ ID NO:47 and SEQ ID NO:48 may also be functionally defined as sequences which are capable of hybridizing to a nucleic acid segment containing the complement of SEQ ID NO:47 and SEQ ID NO:48, respectively, under relatively stringent conditions. Suitable relatively stringent hybridization conditions will be well known to those of sill in the art (Sambrook et al, 1989).
It is also important to understand the molecular events which lead to progression from pre-invasive to invasive breast cancer. Breast cancer is a disease that is presumed to involve a series of genetic alterations that confer increasing growth independence and metastatic capability on somatic cells. Identifying the molecular events that lead to the initial development of a neoplasm is therefore critical to understanding the fundamental mechanisms by which tumors arise and to the selection of optimal targets for gene therapy and chemopreventive agents. As intermediate endpoints in neoplastic development, some pre-malignant breast lesions represent important, and possibly rate-limiting steps in the progression of human breast cancer, and careful epidemiological studies have established the relative risk for breast cancer development for specific histologic lesions. In particular, invasive breast cancer develops in the region of the previous biopsy site in at least 25-30% of patients following diagnosis of non-comedo DCIS providing strong evidence that this pre-malignant lesion is a determinant event in breast cancer progression. While these morphologically defined intermediate endpoints have been widely accepted, progress in defining the molecular correlates of these lesions has been hampered by an inability to identify and sample them in a manner which would allow the application of molecular techniques.
The present invention includes a comparison of gene expression between multiple breast tissue biopsy samples as a means to identify differentially expressed genes in pre-malignant breast disease compared with normal breast tissue. These genetic markers should be extremely useful reagents for early diagnosis of breast cancer, and for the delineation of molecular events in progression of breast cancer.
Identification of gene markers which are expressed in the majority of pre-invasive breast cancer tissue samples involves cDNA library preparation from both normal and abnormal tissue. This is followed by either a modified differential display method or a differential screening method to identify differential expression of genes which is subsequently confirmed by RT-PCR, nuclease protection assays and in situ hybridization of DCIS tissue RNA and control tissue RNAs (Sambrook et al, 1989). Use of genetic engineering methods can bias the screening to specifically identify genes whose encoded proteins are secreted or are present at the cell surface, in order to find proteins which will be useful markers for diagnostic blood tests (secreted proteins) or for diagnostic imaging studies (cell surface proteins).
Thus, the method of the present invention begins with the collection of at least one tissue sample by a microscopically-directed collection step in which a punch biopsy is obtained exclusively from abnormal tissue which exhibits histological or cytological characteristics of pre-invasive breast cancer. Preferably, the sample site will be an isolatable tissue structure, such as ductal epithelial cells from pre-invasive breast cancer tissue. The mRNA is purified from the sample. Then, a cDNA library is prepared from the mRNA purified from the abnormal tissue sample (Sambrook et al, 1989).
A normal tissue sample is then obtained from the patient, using a sample site from an area of tissue which does not exhibit histological or cytological characteristics of pre-invasive cancer. A cDNA library is also prepared from this normal tissue sample.
The abnormal tissue cDNA library can then be compared with the normal tissue cDNA library by differential display or differential screening to determine whether the expression of at least one marker gene in the abnormal tissue sample is different from the expression of the same marker gene in the normal tissue sample.
Further diagnostic steps can be added to the method by cloning the marker gene using sequence-based amplification to create a cloned marker gene which can then be DNA-sequenced in order to derive the protein sequence. The protein sequence is then used to generate antibodies which will recognize these proteins by antibody recognition of the antigen. The presence of the antibody-recognized antigen can then be detected by means of conventional medical diagnostic tests.
This invention also includes methods of screening for compounds and gene therapy methods using the BRCA1 gene. BRCA1 mRNA is expressed at 5-10 fold higher levels in normal mammary tissue than in invasive breast cancer samples. Having demonstrated that mRNA expression levels of BRCA1 are higher in normal mammary cells than in cancer cells, antisense methods were used to test the hypothesis that BRCA1 expression inhibits cell growth. These tests showed that diminished expression of BRCA1 increased the proliferative rate of breast cells.
An object of the present invention, then, is to provide a method of early detection of pre-invasive breast cancer in human tissue.
It is a further object of this invention to identify early marker genes for pre-invasive breast disease which can be used in screening methods for early pre-invasive breast cancer.
It is also an object of this invention to produce a cDNA library from pre-invasive breast cancer tissue resulting in a permanent genetic sample of that pre-invasive breast cancer tissue.
It is also an object of this invention to provide a drug or biological screening method using the BRCA 1 promoter region and gene therapy method using the BRCA 1 gene.